Standards/quality control products are essential for quality control at every stage of molecular diagnostic testing. Regardless of the platform, whether it's NGS, QPCR, or digital PCR, standards are relied upon to evaluate the accuracy and sensitivity of test results.

As precise and stable reference materials with well-defined characteristics (such as known mutation sites and mutation frequencies), standards provide assurance for IVD companies developing test kits, in-house molecular diagnostic laboratories (LDTs), and third-party medical testing laboratories (ICLs). Clinical testing centers also use standards when conducting external quality assessments across their testing centers. Furthermore, pharmaceutical companies also require standards for companion diagnostics during drug development.

CB-Gene's molecular diagnostic standards can be categorized into five application areas:

Oncology: 3S3P1O (Mutaion/Fusion/CNV/Methylation/Panel/Pair/Package/Other);

Pathogens: Common bacteria, viruses, fungi, such as HBV, HPV, and COVID-19;

Drug Metabolism: Folate metabolism, warfarin, tamoxifen, tacrolimus, etc.;

Genetic Diseases: Thalassemia, deafness, SMA, etc.;

Drug R&D: Lentiviral integration site standards, host residue standards, etc.

gDNA: Whole-genome DNA extracted from a cell line with a specific mutation;

RNA: Total RNA extracted from a cell line with a specific mutation;

Cell Pellet: Cell pellet with a specific mutation obtained by centrifugation after cell culture;

ctDNA: DNA fragments of 160 bp ± 10%, simulating patient samples from liquid biopsies;obtained by
① ultrasonic fragmentation of gDNA or
② enzymatic digestion of chromosomes;
ctDNA fragments are available as purified fragments, or ctDNA can be dissolved in artificial plasma at a specific concentration to simulate unpurified clinical plasma samples.

FFPE Block: A specific number of cells with a specific mutation are formalin-fixed and embedded into a paraffin block;

FFPE Slide: An embedded paraffin block cut into individual slides;

Other: Inactivated bacterial solution, inactivated virus, pseudovirus, or specific matrices (such as serum, stool, etc.).

There may be many reasons why the AF test results are not as expected.
There are usually the following common reasons:
1) Differences in the methodological aspects of the technical platform. The AF value and error range presented by COA are based on the results of the absolute quantitative calculation of digital PCR.
When the standard is detected by other methodologies (such as NGS), the deviation of AF may be amplified. This amplification is related to the type of mutation detected, the copy number and sequence specificity of the target gene, the chromosome ploidy of the sample, the specific experimental technical route adopted, and the subsequent bioinformatics analysis method. Specifically, 1) When the mutation is SNV, the AF value obtained by digital PCR and NGS generally has a small deviation, while the AF value deviation of CNV and fusion mutations is large. From an experimental perspective, digital PCR is an amplification and quantification of the complete gDNA sample, while the sample often needs to be sheared during the NGS library construction process. Shearing usually makes it easy for the AF of CNV and fusion mutations to deviate from that before the shearing. We often observe this phenomenon when using digital PCR to detect CNV and fusion mutation samples before and after the shearing. We have also found this difference after the shearing in some samples with abnormal overall chromosome ploidy. In addition, from the perspective of post-test bioinformatics analysis, SNV The calculation and analysis methods for AF values ​​are relatively mature and unified, and are closer to the calculation principles of digital PCR AF values. Therefore, the differences between NGS and digital PCR results are often small. However, the AF calculation of CNV and fusion mutations is more dependent on bioinformatics algorithms. The schemes are less unified and mature, and the algorithms vary greatly from one company to another. This is one of the reasons for the large difference between it and digital PCR calibration AF.

Furthermore, from a methodological perspective, digital PCR is a simple, single-shot PCR method for absolute quantification. NGS methods, whether using amplicon or capture schemes, may involve multiple or multiplex PCR during library construction and sequencing. This PCR process can produce some biased amplification, leading to deviations in AF values. We have also verified through digital PCR experiments that AF values ​​for PCR products vary between pre- and post-library construction for certain samples and loci. Furthermore, insufficient or uneven NGS sequencing depth can also cause AF values ​​to differ from expectations. For example, at sites with high or low GC content, the sequencing depth may be uneven compared to other regions, leading to AF deviations. Furthermore, at sites with high copy number amplification, localized insufficient sequencing depth can result in the upper limit of detection being lower than the true copy number, necessitating a higher sequencing depth to reflect the true copy number.

These are just some common causes of AF deviation. Overall, the causes of AF discrepancies are multifaceted and complex, requiring specific analysis of each case. When encountering actual cases, we may require the client's original data and experimental details to facilitate analysis.

QIAamp DNA FFPE Tissue Kit (Qiagen)

Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific)
Specifications: Extraction yield: >400 ng per two slides with 15 μm/slide

1) Digital PCR primer probes target SNVs or shorter indels (e.g., EGFR 19Del, 20Ins). They typically include a pair of primers and two probes labeled with different fluorophores (e.g., FAM/VIC, FAM/HEX, etc.) for identifying mutant and wild-type copy numbers.

2) Digital PCR primer probes target CNVs. They typically include two pairs of primers and two probes labeled with different fluorophores (e.g., FAM/VIC, FAM/HEX, etc.) for amplifying and quantifying the copy number of the target gene and the reference gene, respectively. (Reference genes are generally selected as relatively conservative genes that are less susceptible to copy number variation, such as RPP30. Although reference genes are relatively conservative and less susceptible to copy number aberrations, this is not absolute. Sometimes, to improve detection accuracy, it is advisable to select two or three different reference genes for comparison.)

3) Digital PCR primer probes target DNA fusion mutations and typically consist of two different primer pairs and two probes labeled with different fluorophores (e.g., FAM/VIC, FAM/HEX, etc.). These are used to amplify, identify, and quantify the target gene's breakpoint copy number and the total target gene copy number, respectively.

4) Digital PCR primer probes target RNA and typically consist of a primer pair and a probe labeled with a fluorophore. These are used to amplify, identify, and quantify the target gene's copy number.